Recently, a large number of proteins, polypeptides, synthetic compounds, and compounds extracted from natural resources having physiological activity have been found out and the application thereof to pharmaceuticals has been extensively studied. However, these physiologically active substances have short half-lives in blood when they are injected to a body and hence it is difficult to obtain a sufficient pharmacological effect. This is because the physiologically active substances injected to a body are usually cleared from the body because of the filtration through glomeruli in the kidney and the uptake by macrophages in the liver, spleen, and the like. Therefore, it is attempted to improve the behavior in a body by including these physiologically active substances in liposomes or polymer micelles or increasing their molecular weight through chemical modification with polyethylene glycol which is an amphiphatic polymer. Polyethylene glycol exhibits a low interaction with the other bio-components owing to its steric repulsion effect and as a result, proteins and polypeptides such as enzymes modified with polyethylene glycol exhibit an effect of avoiding the filtration through glomeruli in the kidney and bio-reactions such as immunoreaction, so that they achieve half-lives in blood longer than those of unmodified substances. Moreover, they also have decreased toxicity and antigpnicity and further exhibit an effect of enhancing the solubility of a sparingly water-soluble compound having a high hydrophobicity.
In the case of modifying a physiologically active substance with a linear polyethylene glycol, it is known that modification effects such as half-lives in blood is enhanced as molecular weight of the polyethylene glycol increases. Therefore, synthesis of a reactive polyethylene glycol derivative having a higher molecular weight has been investigated. However, when a starting methoxypolyethylene glycol is synthesized by addition polymerization of ethylene oxide, particularly in the case that a high-molecular-weight one is intended to obtain, there is increased side-reaction such as vinyl etherification and impurities such as compounds having a two-time molecular weight derived from an initiator. Accordingly, it is difficult to obtain a methoxypolyethylene glycol having a high purity and a high molecular weight. As a result, it is difficult to obtain a reactive polyalkylene glycol derivative having a high purity and a high molecular weight, which is suitable for modification of pharmaceuticals.
Moreover, when a peptide or drug is modified with many polyethylene glycol molecules for obtaining a sufficient effect of the modification with polyethylene glycol, the active site of the peptide or drug is blocked and hence there may arise problems that its own function and efficacy cannot be exhibited sufficiently and enough solubility in water cannot be obtained.
For solving such problems, an attempt to solve such problems using a branched polyethylene glycol derivative has been made. By using a branched one, it becomes possible to introduce two highly pure polyethylene glycol chains per one modifying point and hence modification with a polyethylene glycol chain having a high molecular weight is enabled. Moreover, it becomes possible to cover larger surface of a physiologically active substance with a polyethylene glycol chain. Furthermore, it becomes possible to reduce modification points as compared with the case of modification with a linear one, which enables inhibition of decrease in activity of the physiologically active substance.
JP-B-61-42558 proposes.a polyethylene glycol-modified L-asparaginase. Cyanuric chloride as a starting material for a branched reactive polyethylene glycol derivative used in the publication has three reactive sites and hence it is difficult to introduce two polyethylene glycol chains thereinto selectively. Accordingly, it is difficult to synthesize a highly pure polyethylene glycol-modified L-asparaginase.
Also, JP-A-10-67800 proposes a polyethylene glycol-modified interferon α. The branched reactive polyethylene glycol derivative used in the publication has lysine as a skeleton, wherein methoxypolyethylene glycols are combined with two amino groups of the lysine and then the carboxyl residue is converted into a succinimide ester group. In the case of the branched polyethylene glycol having a lysine skeleton, a reactive functional group is present near to the lysine skeleton and hence there is a possibility that conversion of the reaction with a physiologically active substance decreases due to steric hindrance of the own polyethylene glycol chains. Particularly, in the case that a site to be modified is present inside the polypeptide, there is a possibility that reaction is difficult to proceed in a polyethylene glycol derivative wherein a reactive group is present near to the skeleton.
Accordingly, it is desired to develop a highly reactive branched polyalkylene glycol derivative which solves the problems of these conventional polyethylene glycol derivatives, and a modified bio-related substance.